The cytoskeleton plays a critical role in many developmental processes, from cell motility to asymmetric segregation of cellular components. The recent isolation of a C. elegans mutant lacking a functional class VI myosin (spe-15) highlights the importance of this actin-based motor protein in asymmetric organelle segregation during development. Worms with a deletion in spe-15 are sterile and exhibit spermatid defects due to improper organelle partitioning (Kelleher et al. 2000, submitted). The molecular explanation for this mutant phenotype has not been elucidated, but could offer further insight into roles for the cytoskeleton and unconventional myosins in developmental processes. This proposal encompasses two major objectives. The first objective is to define the possible roles for myosin VI in asymmetric organelle partitioning during spermatogenesis in C, elegans. GFP-SPE-15 fusion constructs will be used to rescue spe-15 in myosin VI deletion animals, as well as to study SPE-15 localization over time in developing spermatocytes and co- localization of SPE-15 with organelles. The second objective is to further analyze the structure and function of myosin VI. Mutated forms of SPE- 15 will be tested for their ability to rescue spe-15 and to co-localize with organelles. Noncomplementation screens will be performed to isolate new spe-15 alleles, including temperature sensitive mutants. A yeast two-hybrid screen will be used to identify potential myosin VI cargo docking proteins. The vast genetic knowledge available (including the genome sequence), short life cycle, small size, body transparency, self- fertilization, and ease of genetic manipulation make C. elegans the system of choice for these studies.